Griffitts:Common buffers: Difference between revisions
(35 intermediate revisions by 4 users not shown) | |||
Line 1: | Line 1: | ||
<div class="noprint"> | |||
{{Template:Griffitts}} | |||
</div> | |||
==Electrophoresis Buffers== | ==Electrophoresis Buffers== | ||
===2% bromophenol blue=== | |||
* 0.4 g bromophenol blue (BPB) | |||
* 20 mL 50 mM [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|Tris pH 8.0]]<br> | |||
Sterile filter | |||
===8X DNA loading=== | ===8X DNA loading=== | ||
* 40% [[ | * 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]] | ||
* 1/50 dilution of [[ | * 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]] | ||
===6X SDS loading=== | ===6X SDS loading=== | ||
* 7.5 mL | * 7.5 mL [[Griffitts:Stock solutions#80% Glycerol (75 mL)|80% glycerol]] | ||
* 2.5 mL | * 2.5 mL [[Griffitts:Common buffers#1 M Tris, pH 6.8 (75 mL)|1 M Tris pH 6.8]] | ||
* 1.2 g | * 1.2 g SDS | ||
* 600 μL | * 600 μL 2-mercaptoethanol (in fume hood) | ||
* 150 μL | * 150 μL [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]] | ||
===6X NAT loading=== | |||
=== | * 7.5 mL [[Griffitts:Stock solutions#80% Glycerol (75 mL)|80% glycerol]] | ||
* | * 2.5 mL [[Griffitts:Common buffers#1 M Tris, pH 6.8 (75 mL)|1 M Tris pH 6.8]] | ||
* 5 μL 2-mercaptoethanol (in fume hood) | |||
* 150 μL [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]] | |||
===10X NAT running=== | |||
* 3 g Tris Base | |||
* 14.4 g glycine | |||
* dH<sub>2</sub>O to 100 mL | |||
===DNA ladder=== | ===DNA ladder=== | ||
* 147 μL ddH<sub>2</sub>0 | * 147 μL ddH<sub>2</sub>0 | ||
* 34 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading]] buffer | * 34 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading]] buffer | ||
* 20 μL 1 kb ladder from NEB | * 20 μL 1 kb ladder from NEB | ||
* 2 μL | * 2 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|0.25 M EDTA]]<br> | ||
Note: use 5 μL per lane | Note: use 5 μL per lane; 3kb band = 60 ng | ||
===Protein ladder=== | ===Protein ladder=== | ||
* 1 mL ddH<sub>2</sub>0 | * Open vial of Sigma SDS6H2-1VL | ||
* 500 μL [[Griffitts:Common buffers#6X SDS loading|6X SDS loading]] | * Add 1 mL ddH<sub>2</sub>0 | ||
* | * Add 500 μL [[Griffitts:Common buffers#6X SDS loading|6X SDS loading]] | ||
* Aliquot 50 μL per tube and store at -20°C<br> | |||
Note: use 5 μL per lane | Note: use 5 μL per lane | ||
=== | ===ethidium bromide (10 mL)=== | ||
* 10 mL ddH<sub>2</sub>0 | * 10 mL ddH<sub>2</sub>0 | ||
* 10 mg | * 10 mg ethidium bromide<br> | ||
Note: use 8 μL per 50 mL gel | Note: use 8 μL per 50 mL gel | ||
===50X | ===50X TAE (500 mL)=== | ||
* | * 400 mL ddH<sub>2</sub>0 | ||
* | * 121 g Trizma base | ||
* | * 28 mL glacial acetic acid | ||
* | * 4 g Na<sub>2</sub>EDTA<br> | ||
Stir until EDTA is dissolved.<br> | |||
Note: this is half the amount of | Final pH should be between 8.1 and 8.4<br> | ||
===10X Laemmli running buffer=== | Note: this is half the amount of EDTA compared to standard TAE. | ||
* 30.3 g | |||
* 144.2 g | ===10X Laemmli running buffer (1 L)=== | ||
* 10 g [[ | * 30.3 g Trizma base (Tris) | ||
* 144.2 g glycine | |||
* 10 g sodium dodecyl sulfate ([[SDS]]) | |||
QS to volume with ddH<sub>2</sub>0 | QS to volume with ddH<sub>2</sub>0 | ||
=== | |||
* 120 mL | ===Coomassie stain (300 mL)=== | ||
* 120 mL ethanol | |||
* 0.3 g Brilliant Blue R<br> | * 0.3 g Brilliant Blue R<br> | ||
Mix for 30 min. | Mix for 30 min. | ||
* 30 mL | * 30 mL acetic acid | ||
* 150 mL ddH<sub>2</sub>0 | * 150 mL ddH<sub>2</sub>0 | ||
===destain solution (1 L)=== | ===Coomassie destain solution (1 L)=== | ||
* 550 mL ddH<sub>2</sub>0 | * 550 mL ddH<sub>2</sub>0 | ||
* 400 mL | * 400 mL ethanol | ||
* 50 mL | * 50 mL acetic acid | ||
==DNA extraction buffers== | ==DNA extraction buffers== | ||
===colony lysis solution (for PCR)=== | ===colony lysis solution (for PCR) (75 mL)=== | ||
* 5 | * 72.15 mL of sterile ddH<sub>2</sub>O (i.e. remove 2.85 mL from a 75-mL bottle of sterile ddH<sub>2</sub>O) | ||
* 2 | * 375 μL of [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] (final concentration: 5 mM) | ||
* 0.5% | * 600 μL of [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] (final concentration: 2 mM) | ||
* 1.875 mL of [[Griffitts:Common buffers#20% Triton X-100|20% Triton X-100]] (final concentration: 0.5%) | |||
** in fridge with the antibiotics<br> | |||
Note: Store in fridge<br> | |||
===TE buffer=== | |||
'''For 75 mL T<sub>3</sub>E<sub>0.3</sub>:''' | |||
* 74.685 mL ddH<sub>2</sub>O (i.e. remove 315 μL from 75 mL ddH<sub>2</sub>O) | |||
* 225 μL [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] | |||
* 90 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] | |||
<font color=green>Note: T<sub>3</sub>E<sub>0.3</sub> is what we use most of the time in the lab</font><br> | |||
'''For 75 mL T<sub>5</sub>E<sub>0.5</sub>:''' | |||
* 74.475 mL ddH<sub>2</sub>O (i.e. remove 525 μL from 75 mL ddH<sub>2</sub>O) | |||
* 375 μL [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] | |||
* 150 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] | |||
'''For 75 mL T<sub>10</sub>E<sub>1</sub>:''' | |||
* 73.95 mL ddH<sub>2</sub>O (i.e. remove 1.05 mL from 75 mL ddH<sub>2</sub>O) | |||
* 750 μL [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] | |||
* 300 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] | |||
'''For 75 mL T<sub>50</sub>E<sub>10</sub>:''' | |||
* 68.25 mL ddH<sub>2</sub>O (i.e. remove 6.75 mL from 75 mL ddH<sub>2</sub>O) | |||
* 3.75 mL [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] | |||
* 3 mL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] | |||
===1 M Tris, pH 6.8 (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 9.09 g Tris (Trizma base) | |||
* pH to 6.8 with HCl | |||
Autoclave | |||
===1 M Tris, pH 7.5 (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 9.09 g Tris (Trizma base) | |||
* pH to 7.5 with HCl | |||
Autoclave | |||
===1 M Tris, pH 8.0 (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 9.09 g Tris (Trizma) | |||
* pH to 8.0 with HCl | |||
Autoclave | |||
===1 M Tris, pH 8.8 (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 9.09 g Tris (Trizma) | |||
* pH to 8.8 with HCl | |||
Autoclave | |||
===0.25 M EDTA (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 6.98 g EDTA | |||
* pH to 8.0 with NaOH | |||
Autoclave | |||
===10% Triton X-100 (40 mL)=== | |||
* 36 mL dH<sub>2</sub>O | |||
* 4 mL Triton X-100 | |||
Note: Stock solution of Triton X-100 is very viscous, so dispense slowly<br> | |||
Note: do not filter sterilize<br> | |||
Store at 4°C | |||
===20% Triton X-100 (40 mL)=== | |||
* 32 mL dH<sub>2</sub>O | |||
* 8 mL Triton X-100 | |||
Note: Stock solution of Triton X-100 is very viscous, so dispense slowly<br> | |||
Note: do not filter sterilize<br> | |||
Store at 4°C | |||
===0.5 M MOPS (40 mL)=== | |||
* 20 mL ddH<sub>2</sub>O | |||
* 4.19 g MOPS | |||
* pH to 7.0 (~3.5 mL NaOH) | |||
QS to volume with ddH<sub>2</sub>O | |||
==plasmid purification solutions== | ==plasmid purification solutions== | ||
=== | ===Resuspension Buffer=== | ||
* | * 68.25 mL ddH<sub>2</sub>O (i.e. remove 6.75 mL from 75 mL ddH<sub>2</sub>O) | ||
* | * 3.75 mL [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] | ||
* 3 mL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] | |||
===lysis solution=== | ===lysis solution=== | ||
* 1% [[ | * 1% [[Griffitts:Stock solutions#20% SDS (75 mL)|SDS]] | ||
* 200 | * 200 mM NaOH | ||
=== | ===Neutralization solution=== | ||
* 4 | * 4 M potassium acetate | ||
=== | * pH to 5.5 with acetic acid | ||
* | |||
* | ===RNAse A=== | ||
* 20 mg/mL RNAse A (in freezer) | |||
* 10 mM [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|Tris pH 8.0]] | |||
Note: RNAse A is purchased from Sigma (R-6513) | |||
===silica suspension=== | ===silica suspension=== | ||
Wash 5X with ddH<sub>2</sub>0<br> | * Wash 5X with ddH<sub>2</sub>0<br> | ||
Resuspend as 50% slurry in ddH<sub>2</sub>0 | * Resuspend as 50% slurry in ddH<sub>2</sub>0<br> | ||
Store at -20°C | |||
===silica wash solution=== | ===silica wash solution=== | ||
* 50% | * 50% ethanol | ||
* 10 | * 10 mM [[Griffitts:Common buffers#1 M Tris, pH 7.5 (75 mL)|Tris pH 7.5]] | ||
* 50 | * 50 mM [[Griffitts:Stock solutions#5 M NaCl (75 mL)|NaCl]] | ||
* 2.5 | * 2.5 mM [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|EDTA]] | ||
===Proteinase K=== | |||
* 20 mg/mL Proteinase K (in freezer) | |||
* 20 mM [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|Tris pH 8.0]] | |||
Store at -20°C | |||
===Lysozyme=== | |||
* 1 mL dH<sub>2</sub>O | |||
* 100mg lysozyme (in freezer) | |||
Store at -20°C | |||
==Bases== | |||
===2N KOH (40 mL)=== | |||
* 4.49 g KOH pellets | |||
* 40 mL ddH<sub>2</sub>O | |||
NOTE: for KOH 2N = 2M | |||
===2N NaOH (40 mL)=== | |||
* 3.2 g NaOH pellets | |||
* 40 mL ddH<sub>2</sub>O | |||
NOTE: for NaOH 2N = 2M |
Latest revision as of 14:42, 13 December 2013
Electrophoresis Buffers
2% bromophenol blue
- 0.4 g bromophenol blue (BPB)
- 20 mL 50 mM Tris pH 8.0
Sterile filter
8X DNA loading
- 40% glycerol
- 1/50 dilution of 2% bromophenol blue
6X SDS loading
- 7.5 mL 80% glycerol
- 2.5 mL 1 M Tris pH 6.8
- 1.2 g SDS
- 600 μL 2-mercaptoethanol (in fume hood)
- 150 μL 2% bromophenol blue
6X NAT loading
- 7.5 mL 80% glycerol
- 2.5 mL 1 M Tris pH 6.8
- 5 μL 2-mercaptoethanol (in fume hood)
- 150 μL 2% bromophenol blue
10X NAT running
- 3 g Tris Base
- 14.4 g glycine
- dH2O to 100 mL
DNA ladder
- 147 μL ddH20
- 34 μL 8X DNA loading buffer
- 20 μL 1 kb ladder from NEB
- 2 μL 0.25 M EDTA
Note: use 5 μL per lane; 3kb band = 60 ng
Protein ladder
- Open vial of Sigma SDS6H2-1VL
- Add 1 mL ddH20
- Add 500 μL 6X SDS loading
- Aliquot 50 μL per tube and store at -20°C
Note: use 5 μL per lane
ethidium bromide (10 mL)
- 10 mL ddH20
- 10 mg ethidium bromide
Note: use 8 μL per 50 mL gel
50X TAE (500 mL)
- 400 mL ddH20
- 121 g Trizma base
- 28 mL glacial acetic acid
- 4 g Na2EDTA
Stir until EDTA is dissolved.
Final pH should be between 8.1 and 8.4
Note: this is half the amount of EDTA compared to standard TAE.
10X Laemmli running buffer (1 L)
- 30.3 g Trizma base (Tris)
- 144.2 g glycine
- 10 g sodium dodecyl sulfate (SDS)
QS to volume with ddH20
Coomassie stain (300 mL)
- 120 mL ethanol
- 0.3 g Brilliant Blue R
Mix for 30 min.
- 30 mL acetic acid
- 150 mL ddH20
Coomassie destain solution (1 L)
- 550 mL ddH20
- 400 mL ethanol
- 50 mL acetic acid
DNA extraction buffers
colony lysis solution (for PCR) (75 mL)
- 72.15 mL of sterile ddH2O (i.e. remove 2.85 mL from a 75-mL bottle of sterile ddH2O)
- 375 μL of 1 M Tris pH 8.0 (final concentration: 5 mM)
- 600 μL of .25 M EDTA (final concentration: 2 mM)
- 1.875 mL of 20% Triton X-100 (final concentration: 0.5%)
- in fridge with the antibiotics
- in fridge with the antibiotics
Note: Store in fridge
TE buffer
For 75 mL T3E0.3:
- 74.685 mL ddH2O (i.e. remove 315 μL from 75 mL ddH2O)
- 225 μL 1 M Tris pH 8.0
- 90 μL .25 M EDTA
Note: T3E0.3 is what we use most of the time in the lab
For 75 mL T5E0.5:
- 74.475 mL ddH2O (i.e. remove 525 μL from 75 mL ddH2O)
- 375 μL 1 M Tris pH 8.0
- 150 μL .25 M EDTA
For 75 mL T10E1:
- 73.95 mL ddH2O (i.e. remove 1.05 mL from 75 mL ddH2O)
- 750 μL 1 M Tris pH 8.0
- 300 μL .25 M EDTA
For 75 mL T50E10:
- 68.25 mL ddH2O (i.e. remove 6.75 mL from 75 mL ddH2O)
- 3.75 mL 1 M Tris pH 8.0
- 3 mL .25 M EDTA
1 M Tris, pH 6.8 (75 mL)
- 75 mL ddH2O
- 9.09 g Tris (Trizma base)
- pH to 6.8 with HCl
Autoclave
1 M Tris, pH 7.5 (75 mL)
- 75 mL ddH2O
- 9.09 g Tris (Trizma base)
- pH to 7.5 with HCl
Autoclave
1 M Tris, pH 8.0 (75 mL)
- 75 mL ddH2O
- 9.09 g Tris (Trizma)
- pH to 8.0 with HCl
Autoclave
1 M Tris, pH 8.8 (75 mL)
- 75 mL ddH2O
- 9.09 g Tris (Trizma)
- pH to 8.8 with HCl
Autoclave
0.25 M EDTA (75 mL)
- 75 mL ddH2O
- 6.98 g EDTA
- pH to 8.0 with NaOH
Autoclave
10% Triton X-100 (40 mL)
- 36 mL dH2O
- 4 mL Triton X-100
Note: Stock solution of Triton X-100 is very viscous, so dispense slowly
Note: do not filter sterilize
Store at 4°C
20% Triton X-100 (40 mL)
- 32 mL dH2O
- 8 mL Triton X-100
Note: Stock solution of Triton X-100 is very viscous, so dispense slowly
Note: do not filter sterilize
Store at 4°C
0.5 M MOPS (40 mL)
- 20 mL ddH2O
- 4.19 g MOPS
- pH to 7.0 (~3.5 mL NaOH)
QS to volume with ddH2O
plasmid purification solutions
Resuspension Buffer
- 68.25 mL ddH2O (i.e. remove 6.75 mL from 75 mL ddH2O)
- 3.75 mL 1 M Tris pH 8.0
- 3 mL .25 M EDTA
lysis solution
- 1% SDS
- 200 mM NaOH
Neutralization solution
- 4 M potassium acetate
- pH to 5.5 with acetic acid
RNAse A
- 20 mg/mL RNAse A (in freezer)
- 10 mM Tris pH 8.0
Note: RNAse A is purchased from Sigma (R-6513)
silica suspension
- Wash 5X with ddH20
- Resuspend as 50% slurry in ddH20
Store at -20°C
silica wash solution
- 50% ethanol
- 10 mM Tris pH 7.5
- 50 mM NaCl
- 2.5 mM EDTA
Proteinase K
- 20 mg/mL Proteinase K (in freezer)
- 20 mM Tris pH 8.0
Store at -20°C
Lysozyme
- 1 mL dH2O
- 100mg lysozyme (in freezer)
Store at -20°C
Bases
2N KOH (40 mL)
- 4.49 g KOH pellets
- 40 mL ddH2O
NOTE: for KOH 2N = 2M
2N NaOH (40 mL)
- 3.2 g NaOH pellets
- 40 mL ddH2O
NOTE: for NaOH 2N = 2M